To elucidate the mechanism of the effect of biochar on the efficient nitrogen removal of anammox systems, this study analyzed the actual and theoretical electron transfer requirements and microbial dynamics of the system at varying NO2–N/NH4+- N (1.32-0.6). Results indicated that biochar increased nitrogen removal in RBC by 27 %-30 %, primarily through enhanced partial denitrification compared to the RCK. Under low NO2- – N/NH4+- N (0.6-0.8), an excess of ammonium cooperates with the biochar-mediated partial denitrification (PD) process, achieving a nitrogen removal of 85 %-90 %, with biochar contributing 26.4 %. Despite having a lower electron transfer capacity (1.13 mol e-) than the system’s electron demand (1.66 mol e-), biochar increased electron transfer activity by 33.6-41.9 %. It achieved this by either triggering extracellular polymeric substances (EPS) secretion to promote electron transfer, or directly supplying additional electrons. Metagenomic differences revealed that the combination of the biochar biofilm and suspended sludge system regulated the competitive relationship between AnAOB and denitrifying bacteria. The abundance of Candidatus Brocadia on the biochar biofilm increased from 0.79 % in phase I to 33.25 % in phase IV. Furthermore, the addition of biochar facilitated the expression of functional genes related to anammox and denitrification processes. These findings provide a novel strategy for more efficient and environmentally friendly applications of anammox process with reduced energy consumption.
